Our laboratory has been focused on the development of a synthetic bone graft substitute (BGS) for the past 15 years. Our experimental bone graft substitutes have included PLGA- (Poly-lactide-co-glycolide) and collagen-based scaffolds seeded with autologous bone marrow stromal cells (BMSC), and stimulated by osteoinductive agents including BMP-2. With these BGS models, we have generated new bone both in vitro, using a 3-dimensional tissue culture system, and in vivo, resulting in the healing of critical-sized cranial defects in the rabbit. While our studies have been promising, a clinically useful BGS remains elusive. The carrier is of critical importance in the development of a BGS. PLGA has shown much promise, being both biocompatible and biodegradable. However, there are limitations to the use of PLGA in tissue engineering. PLGA induces the formation of acidic degradation products that can affect local PH, possibly inducing inflammation. Collagen based scaffolds, including collagen-GAG, have been widely used in bone tissue engineering because collagen is a naturally existing component of extracellular matrix that promotes cell binding, exhibits low antigenicity and has excellent haemostatic property. We demonstrated that human mesenchymal stem cells (MSC) seeded in collagen scaffolds exhibit accelerated and robust mineralization and bone formation in comparison to their counterparts seeded in PLGA scaffolds. However, we also discovered in our in vitro studies that cell-seeded collagen scaffolds undergo significant contraction compared to PLGA scaffolds. The contraction can greatly hamper application of collagen implants in repairing bony defects. In order to limit volume loss and destruction of collagen implants, we propose to stabilize the structure of collagen scaffolds by incorporating nanoparticulate calcium phosphate (CaP) on the collagen-GAG fibrils. Our initial test results indicate using MC-GAG scaffolds can greatly prevent contraction caused by adhesion and differentiation of hMSCs. It is also superior to collagen-GAG in supporting new bone formation. Our current proposal intends to further investigate the feasibility of MC-GAG scaffolds in bone tissue engineering. In this proposal, we will compare collagen-GAG and MC-GAG scaffolds for their ability to support osteogenic differentiation in rabbit BMSCs cultured in vitro. We will also compare the efficacy of cellular scaffolds made of collagen-GAG and MC-GAG in healing a critical-sized rabbit cranial defect by performing histological and mechanical analysis of newly formed bone. Finally, we will study integrins and their downstream signaling molecules responsible for the differences between collagen-GAG and MC-GAG in their ability to control contraction and support osteogenic differentiation in rBMSCs. We expect that MC-GAG scaffolds will (1) enhance osteogenic differentiation and reduce contraction in rabbit BMSCs compared to collagen alone, and (2) accelerate new bone formation in a rabbit cranial defect model compared to collagen alone. We anticipate that changes in integrin signaling are responsible for enhanced differentiation and reduced contraction of rBMSCs in MC-GAG scaffolds.